Project Summary Influenza is a major cause of morbidity and mortality worldwide, with seasonal epidemics causing an estimated 290,000 to 650,000 deaths every year. Annual vaccine efficacy can be as low as 10% due to antigenic mismatch between circulating influenza strains and vaccine strains, underscoring the need for a universal influenza virus vaccine. Our lab has developed a novel vaccination strategy utilizing chimeric hemagglutinin (HA) constructs to shift the host immune response from the antigenically variable HA head domain to the conserved HA stalk. Vaccination with live attenuated influenza virus vaccines (LAIVs) expressing these constructs elicits broad protection against different influenza virus strains in animal models. However, concern regarding the reassortment potential of LAIVs with wild-type circulating viruses impedes consideration of wide- scale vaccination with LAIVs containing novel HAs and confines clinical trials with these vaccines to containment units. Previous work from our lab has provided evidence that packaging signals located at the 3' and 5' termini of the viral genomic RNA segments can be utilized to control influenza virus reassortment. By swapping the packaging signals of two segments, we prevented free reassortment of those segments. Based on these data, I have generated more viruses with additional rewired packaging signals to define their reassortment potential using coinfection studies under the hypothesis that they will be reassortment-deficient. To better understand the mechanistic underpinnings of selective packaging, I will perform transposon mutagenesis on the HA vRNA segment and map the specific sequences implicated in key RNA-RNA packaging interactions. The ultimate goal of this project is to establish a novel platform for the development of LAIVs that are unable to freely reassort and gain an understanding of the nucleotide interactions at play in influenza genome packaging and reassortment. This would allow us to create LAIV candidates that contain HAs novel to the human population for either universal or prepandemic vaccination purposes.